- Email: [email protected]
Quantifying Current Events Identifies a Novel Endurance Regulator
Pursuing these findings, the authors describe an elegant molecular mechanism by which HAdV hijacks IFN signalling to repress transcription of E1A, the first viral gene expressed during infection. IFNs dramatically slow the progress of infection by directly repressing expression of the immediate early E1A gene, whose protein is essential for activating expression of the other HAdV early genes and reprogramming the cell into a state conducive to infection [9]. This establishes conditions leading to a persistent HAdV infection in cultures of normal human cells, which steadily produce intermediate amounts of infectious virus over a time frame exceeding 100 days, with limited cytopathic effect [6]. Extensive mutagenesis identified a cisacting repressive element within the E1A enhancer necessary for establishing persistence in response to IFN. Using chromatin immunoprecipitation, IFN treatment was shown to cause recruitment of repressor complexes comprising the Rb family of tumor suppressors and associated E2F transcription factors to this site. This occupancy was also correlated to a reduction in binding by the GABP transcription factors at an adjacent regulatory site, which normally induce high levels of E1A expression immediately upon infection. As expected, viruses lacking this repressive element fail to establish a persistent state in response to IFN treatment. Fascinatingly, this genetic element and functional repression by IFN treatment are conserved between distantly related HAdV species, suggesting that this regulation serves an important evolutionary function. Despite a clear role for Rb and family members in IFN mediated repression of E1A expression, the linkage between these two pathways in the establishment of HAdV persistence has not yet been investigated. It is well established that IFN treatment can shift the phosphorylation state of Rb from the inactive
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hyperphosphorylated form to the active, growth-suppressing hypophosphorylated form by downregulating cyclin-dependent kinase (Cdk) activity [10]. This occurs through a variety of mechanisms, including induction of Cdk inhibitors and/or reduced expression or activity of Cdks and their cyclin regulatory units. It will be interesting to determine how this occurs in HAdV persistence, particularly as Zheng et al. [6] report that type I and type II IFNs lead to occupancy of the repressive element by different Rb family members. It also remains to be determined exactly which defects exist in this pathway in tumor cell models, such as the IFNresponsive A549 adenocarcinoma cells typically used in HAdV studies. Furthermore, the ability of E1A to overcome the challenges presented in a growth-arrested cell in contrast to an IFN-treated cell could provide the key to understanding the role of E2F in both lytic and persistent infections.
1 Department of Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada 2 Departments of Oncology and Otolaryngology, University
of Western Ontario, London, Ontario, Canada 3 London Regional Cancer Program and Lawson Health Research Institute, London, Ontario, Canada *Correspondence: [email protected] (J.S. Mymryk). http://dx.doi.org/10.1016/j.tim.2016.02.007 References 1. Rowe, W.P. et al. (1953) Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84, 570–573 2. Zhang, Y. et al. (2010) Modeling adenovirus latency in human lymphocyte cell lines. J. Virol. 84, 8799–8810 3. Lion, T. (2014) Adenovirus infections in immunocompetent and immunocompromised patients. Clin. Microbiol. Rev. 27, 441–462 4. Chahal, J.S. et al. (2012) The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells. PLoS Pathog. 8, e1002853 5. Toth, K. et al. (2015) STAT2 knockout Syrian hamsters support enhanced replication and pathogenicity of human adenovirus, revealing an important role of type I interferon response in viral control. PLoS Pathog. 11, e1005084 6. Zheng, Y. et al. (2016) E2F/Rb family proteins mediate interferon induced repression of adenovirus immediate early transcription to promote persistent viral infection. PLoS Pathog. 12, e1005415 7. Garnett, C.T. et al. (2009) Latent species C adenoviruses in human tonsil tissues. J. Virol. 83, 2417–2428
Overall, these findings identify a remark8. Hendrickx, R. et al. (2014) Innate immunity to adenovirus. able mechanism by which a virus re-tasks Hum. Gene Ther. 25, 265–284 an antiviral cytokine response to establish 9. Pelka, P. et al. (2008) Intrinsic structural disorder in adenovirus E1A: a viral molecular hub linking multiple diverse a persistent infection. Persistence likely processes. J. Virol. 82, 7252–7263 benefits the virus by allowing the continu- 10. Sangfelt, O. et al. (2000) Mechanisms of interferon-induced cell cycle arrest. Front. Biosci. 5, D479–D487 ous production of new progeny at intermediate levels over a vastly extended timeframe. Consequently, the total number of viral progeny produced by persis- Special Issue: Microbial tent infections may exceed the number Endurance produced by acute and self-limiting infections. Furthermore, the substantially reduced level of viral protein produced during a persistent infection will limit viral antigen presentation, potentially avoiding detection and destruction of the infected cell by the immune system. The establishment of this first in vitro model of persistent HAdV infection brings a new level of complexity and understanding to the replicative 1,2 and cycle of this highly prevalent human patho- Theresa C. Henry 1,3, * gen. Besides providing the opportunity to Mark P. Brynildsen further dissect the molecular mechanism involved in establishing persistence, these In nongrowing microbes, proteome studies may also lead to the development turnover is reduced and identificaof drugs to facilitate rapid viral clearance. tion of newly synthesized,
Spotlight
Quantifying Current Events Identifies a Novel Endurance Regulator
low-abundance proteins is challenging. Babin and colleagues recently utilized bio-orthogonal noncanonical amino acid tagging (BONCAT) to identify actively synthesized proteins in nongrowing Pseudomonas aeruginosa, discovering a regulator whose influences range from biofilm formation to secondary metabolism. Poor growth conditions are frequently encountered by bacteria, and cessation of cell division is a common coping mechanism. For example, when faced with nutrient depletion, some bacteria, such as Bacillus subtilis, form spores which can subsist under stressful conditions for long periods of time and reawaken when the environment has become favorable again [1]. Other bacteria, such as Mycobacterium tuberculosis, may lie latent within a host for many years and reactivate when the host's immune system is compromised [2]. In addition, when assaulted with high concentrations of bactericidal antibiotics, bacteria that had ceased growing beforehand are more likely to tolerate the drug than those that were growing at the time of treatment [3]. Further knowledge of nongrowing phenotypic states will improve our understanding of bacterial ecology and enhance our ability to treat latent infections. However, one challenge that stands in the way of gaining such knowledge is the difficulty of distinguishing proteins synthesized by the modest translational capacity of nongrowing cells from the myriad of proteins lingering from previous growth states. In essence, the turnover rate of their proteomes lengthens considerably, which can hinder the identification of newly synthesized proteins that play a role in microbial endurance under stressful conditions. In a recent article published in PNAS [4], Babin and colleagues addressed this challenge by employing bio-orthogonal noncanonical amino acid tagging (BONCAT) [5] to identify actively synthesized proteins in nongrowing P. aeruginosa,
and they discovered a novel transcriptional regulator with a broad array of influence, which they have termed SutA (survival under transitions A). P. aeruginosa is an opportunistic pathogen that is a common cause of pneumonia in cystic fibrosis patients and immunocompromised individuals, a prevalent resident of biofilms on prosthetic devices, and a frequently observed species in chronic wound infections [6]. During infection, P. aeruginosa could encounter anoxic environments within biofilms, where alternative, exogenous electron acceptors, such as nitrate, are absent or in low abundance [7]. Since P. aeruginosa is poorly suited to propagate in such environments, it enters a state where it is nongrowing, but some metabolic and other essential processes remain active [8]. In order to better understand the factors that enable P. aeruginosa to survive such conditions, Babin and colleagues endeavored to identify newly synthesized proteins, which could be at very low abundance, in nongrowing P. aeruginosa using BONCAT, which labels all proteins being actively synthesized with noncanonical amino acids that have useful chemical handles [5].
synthase is expressed [9]. In their study, Babin and colleagues enriched for labeled proteins by reacting protein lysate with the dialkoxydiphenylsilane (DADPS) biotinalkyne probe and incubating with streptavidin resin. After cleavage of the DADPS linker, proteins were eluted and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Applying the above method to anaerobic P. aeruginosa cultures in minimal media with arginine as the sole carbon source, Babin and colleagues discovered a previously uncharacterized protein, SutA [4]. Upon further investigation, SutA was found to contribute to biofilm formation, pyocanin production, and the ability of cells to transition under fluctuating conditions. Deletion of SutA resulted in smaller biofilms and biofilms with altered morphology as compared to those of wild-type, whereas its overexpression resulted in larger biofilms. Removal of SutA also increased pyocanin production, which is linked to P. aeruginosa virulence, whereas increased SutA abundance resulted in decreased pyocanin production. Additionally, competition experiments between wild-type cells and a SutA deletion strain showed that wild-type cells had an advantage when cultures were alternated between aerobic growth in rich media and anaerobic growth in minimal media. Babin and colleagues went on to perform immunoprecipitation assays followed by LC-MS/MS, to find that SutA coprecipitates with several components of the RNA polymerase core enzyme. Furthermore, chromatin IP (ChIP)-sequencing (seq) and RNA-seq experiments demonstrated that SutA works together with RNA polymerase to increase transcription of ribosomal protein and rRNA genes. Genomewide analysis of the data indicated that SutA upregulates genes involved in energy production and maintenance, while it downregulates genes associated with defense mechanisms and motility.
The BONCAT method is performed by pulse-labeling cultures with an amino acid analog, and thus the analog is only incorporated into proteins translated after pulsation. Labeled proteins can be enriched from the rest of the proteome by ligating a probe to the amino acid analog, and then performing chromatography followed by mass spectrometry to identify the labeled proteins. For their study, Babin and colleagues utilized the methionine surrogate L-azidohomoalanine (Aha), which is able to be incorporated into proteins by the wild-type translational machinery. However, it is worth noting that the BONCAT method can be applied in a cell-specific manner by using another methionine analog, azidonorleucine (Anl), which requires a mutant tRNA synthase for incorporation, and therefore, restricts Babin and colleagues’ study not only prolabeling to cells in which the mutant vides novel insight into the ability of
Trends in Microbiology, May 2016, Vol. 24, No. 5
325
P. aeruginosa to endure anaerobic, fermentative conditions, but it also gives an elegant example of how the challenge of low proteome turnover in nongrowing cells can be addressed with BONCAT. Given its versatility, which has been demonstrated by its application to a range of mammalian cells and several prokaryotic species [10], BONCAT is a powerful tool to apply to understand the physiology of bacteria when they are in nongrowing states. Acknowledgments This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (T.C.H.; F30AI114163) and Princeton University (M.P.B.). The content is solely the
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responsibility of the authors and does not necessarily represent the official views of the funding agencies. 1
Department of Molecular Biology, Princeton University,
Princeton, NJ 08544, USA 2 Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA 3 Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA *Correspondence: [email protected] (M.P. Brynildsen). http://dx.doi.org/10.1016/j.tim.2016.02.014 References 1. Errington, J. (2003) Regulation of endospore formation in Bacillus subtilis. Nat. Rev. Microbiol. 1, 117–126 2. Smith, I. (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16, 463–496 3. Orman, M.A. and Brynildsen, M.P. (2013) Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob. Agents Chemother. 57, 3230–3239
4. Babin, B.M. et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 113, E597– E605 5. Dieterich, D.C. et al. (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. U.S.A. 103, 9482–9487 6. Rybtke, M. et al. (2015) Pseudomonas aeruginosa biofilm infections: community structure, antimicrobial tolerance and immune response. J. Mol. Biol. 427, 3628–3645 7. Schreiber, K. et al. (2006) Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein. J. Bacteriol. 188, 659–668 8. Glasser, N.R. et al. (2014) Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol. Microbiol. 92, 399–412 9. Ngo, J.T. et al. (2009) Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 10. Ngo, J.T. and Tirrell, D.A. (2011) Noncanonical amino acids in the interrogation of cellular protein synthesis. Accounts Chem. Res. 44, 677–685
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hyperphosphorylated form to the active, growth-suppressing hypophosphorylated form by downregulating cyclin-dependent kinase (Cdk) activity [10]. This occurs through a variety of mechanisms, including induction of Cdk inhibitors and/or reduced expression or activity of Cdks and their cyclin regulatory units. It will be interesting to determine how this occurs in HAdV persistence, particularly as Zheng et al. [6] report that type I and type II IFNs lead to occupancy of the repressive element by different Rb family members. It also remains to be determined exactly which defects exist in this pathway in tumor cell models, such as the IFNresponsive A549 adenocarcinoma cells typically used in HAdV studies. Furthermore, the ability of E1A to overcome the challenges presented in a growth-arrested cell in contrast to an IFN-treated cell could provide the key to understanding the role of E2F in both lytic and persistent infections.
1 Department of Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada 2 Departments of Oncology and Otolaryngology, University
of Western Ontario, London, Ontario, Canada 3 London Regional Cancer Program and Lawson Health Research Institute, London, Ontario, Canada *Correspondence: [email protected] (J.S. Mymryk). http://dx.doi.org/10.1016/j.tim.2016.02.007 References 1. Rowe, W.P. et al. (1953) Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84, 570–573 2. Zhang, Y. et al. (2010) Modeling adenovirus latency in human lymphocyte cell lines. J. Virol. 84, 8799–8810 3. Lion, T. (2014) Adenovirus infections in immunocompetent and immunocompromised patients. Clin. Microbiol. Rev. 27, 441–462 4. Chahal, J.S. et al. (2012) The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells. PLoS Pathog. 8, e1002853 5. Toth, K. et al. (2015) STAT2 knockout Syrian hamsters support enhanced replication and pathogenicity of human adenovirus, revealing an important role of type I interferon response in viral control. PLoS Pathog. 11, e1005084 6. Zheng, Y. et al. (2016) E2F/Rb family proteins mediate interferon induced repression of adenovirus immediate early transcription to promote persistent viral infection. PLoS Pathog. 12, e1005415 7. Garnett, C.T. et al. (2009) Latent species C adenoviruses in human tonsil tissues. J. Virol. 83, 2417–2428
Overall, these findings identify a remark8. Hendrickx, R. et al. (2014) Innate immunity to adenovirus. able mechanism by which a virus re-tasks Hum. Gene Ther. 25, 265–284 an antiviral cytokine response to establish 9. Pelka, P. et al. (2008) Intrinsic structural disorder in adenovirus E1A: a viral molecular hub linking multiple diverse a persistent infection. Persistence likely processes. J. Virol. 82, 7252–7263 benefits the virus by allowing the continu- 10. Sangfelt, O. et al. (2000) Mechanisms of interferon-induced cell cycle arrest. Front. Biosci. 5, D479–D487 ous production of new progeny at intermediate levels over a vastly extended timeframe. Consequently, the total number of viral progeny produced by persis- Special Issue: Microbial tent infections may exceed the number Endurance produced by acute and self-limiting infections. Furthermore, the substantially reduced level of viral protein produced during a persistent infection will limit viral antigen presentation, potentially avoiding detection and destruction of the infected cell by the immune system. The establishment of this first in vitro model of persistent HAdV infection brings a new level of complexity and understanding to the replicative 1,2 and cycle of this highly prevalent human patho- Theresa C. Henry 1,3, * gen. Besides providing the opportunity to Mark P. Brynildsen further dissect the molecular mechanism involved in establishing persistence, these In nongrowing microbes, proteome studies may also lead to the development turnover is reduced and identificaof drugs to facilitate rapid viral clearance. tion of newly synthesized,
Spotlight
Quantifying Current Events Identifies a Novel Endurance Regulator
low-abundance proteins is challenging. Babin and colleagues recently utilized bio-orthogonal noncanonical amino acid tagging (BONCAT) to identify actively synthesized proteins in nongrowing Pseudomonas aeruginosa, discovering a regulator whose influences range from biofilm formation to secondary metabolism. Poor growth conditions are frequently encountered by bacteria, and cessation of cell division is a common coping mechanism. For example, when faced with nutrient depletion, some bacteria, such as Bacillus subtilis, form spores which can subsist under stressful conditions for long periods of time and reawaken when the environment has become favorable again [1]. Other bacteria, such as Mycobacterium tuberculosis, may lie latent within a host for many years and reactivate when the host's immune system is compromised [2]. In addition, when assaulted with high concentrations of bactericidal antibiotics, bacteria that had ceased growing beforehand are more likely to tolerate the drug than those that were growing at the time of treatment [3]. Further knowledge of nongrowing phenotypic states will improve our understanding of bacterial ecology and enhance our ability to treat latent infections. However, one challenge that stands in the way of gaining such knowledge is the difficulty of distinguishing proteins synthesized by the modest translational capacity of nongrowing cells from the myriad of proteins lingering from previous growth states. In essence, the turnover rate of their proteomes lengthens considerably, which can hinder the identification of newly synthesized proteins that play a role in microbial endurance under stressful conditions. In a recent article published in PNAS [4], Babin and colleagues addressed this challenge by employing bio-orthogonal noncanonical amino acid tagging (BONCAT) [5] to identify actively synthesized proteins in nongrowing P. aeruginosa,
and they discovered a novel transcriptional regulator with a broad array of influence, which they have termed SutA (survival under transitions A). P. aeruginosa is an opportunistic pathogen that is a common cause of pneumonia in cystic fibrosis patients and immunocompromised individuals, a prevalent resident of biofilms on prosthetic devices, and a frequently observed species in chronic wound infections [6]. During infection, P. aeruginosa could encounter anoxic environments within biofilms, where alternative, exogenous electron acceptors, such as nitrate, are absent or in low abundance [7]. Since P. aeruginosa is poorly suited to propagate in such environments, it enters a state where it is nongrowing, but some metabolic and other essential processes remain active [8]. In order to better understand the factors that enable P. aeruginosa to survive such conditions, Babin and colleagues endeavored to identify newly synthesized proteins, which could be at very low abundance, in nongrowing P. aeruginosa using BONCAT, which labels all proteins being actively synthesized with noncanonical amino acids that have useful chemical handles [5].
synthase is expressed [9]. In their study, Babin and colleagues enriched for labeled proteins by reacting protein lysate with the dialkoxydiphenylsilane (DADPS) biotinalkyne probe and incubating with streptavidin resin. After cleavage of the DADPS linker, proteins were eluted and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Applying the above method to anaerobic P. aeruginosa cultures in minimal media with arginine as the sole carbon source, Babin and colleagues discovered a previously uncharacterized protein, SutA [4]. Upon further investigation, SutA was found to contribute to biofilm formation, pyocanin production, and the ability of cells to transition under fluctuating conditions. Deletion of SutA resulted in smaller biofilms and biofilms with altered morphology as compared to those of wild-type, whereas its overexpression resulted in larger biofilms. Removal of SutA also increased pyocanin production, which is linked to P. aeruginosa virulence, whereas increased SutA abundance resulted in decreased pyocanin production. Additionally, competition experiments between wild-type cells and a SutA deletion strain showed that wild-type cells had an advantage when cultures were alternated between aerobic growth in rich media and anaerobic growth in minimal media. Babin and colleagues went on to perform immunoprecipitation assays followed by LC-MS/MS, to find that SutA coprecipitates with several components of the RNA polymerase core enzyme. Furthermore, chromatin IP (ChIP)-sequencing (seq) and RNA-seq experiments demonstrated that SutA works together with RNA polymerase to increase transcription of ribosomal protein and rRNA genes. Genomewide analysis of the data indicated that SutA upregulates genes involved in energy production and maintenance, while it downregulates genes associated with defense mechanisms and motility.
The BONCAT method is performed by pulse-labeling cultures with an amino acid analog, and thus the analog is only incorporated into proteins translated after pulsation. Labeled proteins can be enriched from the rest of the proteome by ligating a probe to the amino acid analog, and then performing chromatography followed by mass spectrometry to identify the labeled proteins. For their study, Babin and colleagues utilized the methionine surrogate L-azidohomoalanine (Aha), which is able to be incorporated into proteins by the wild-type translational machinery. However, it is worth noting that the BONCAT method can be applied in a cell-specific manner by using another methionine analog, azidonorleucine (Anl), which requires a mutant tRNA synthase for incorporation, and therefore, restricts Babin and colleagues’ study not only prolabeling to cells in which the mutant vides novel insight into the ability of
Trends in Microbiology, May 2016, Vol. 24, No. 5
325
P. aeruginosa to endure anaerobic, fermentative conditions, but it also gives an elegant example of how the challenge of low proteome turnover in nongrowing cells can be addressed with BONCAT. Given its versatility, which has been demonstrated by its application to a range of mammalian cells and several prokaryotic species [10], BONCAT is a powerful tool to apply to understand the physiology of bacteria when they are in nongrowing states. Acknowledgments This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (T.C.H.; F30AI114163) and Princeton University (M.P.B.). The content is solely the
326
Trends in Microbiology, May 2016, Vol. 24, No. 5
responsibility of the authors and does not necessarily represent the official views of the funding agencies. 1
Department of Molecular Biology, Princeton University,
Princeton, NJ 08544, USA 2 Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA 3 Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA *Correspondence: [email protected] (M.P. Brynildsen). http://dx.doi.org/10.1016/j.tim.2016.02.014 References 1. Errington, J. (2003) Regulation of endospore formation in Bacillus subtilis. Nat. Rev. Microbiol. 1, 117–126 2. Smith, I. (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16, 463–496 3. Orman, M.A. and Brynildsen, M.P. (2013) Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob. Agents Chemother. 57, 3230–3239
4. Babin, B.M. et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 113, E597– E605 5. Dieterich, D.C. et al. (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. U.S.A. 103, 9482–9487 6. Rybtke, M. et al. (2015) Pseudomonas aeruginosa biofilm infections: community structure, antimicrobial tolerance and immune response. J. Mol. Biol. 427, 3628–3645 7. Schreiber, K. et al. (2006) Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein. J. Bacteriol. 188, 659–668 8. Glasser, N.R. et al. (2014) Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol. Microbiol. 92, 399–412 9. Ngo, J.T. et al. (2009) Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 10. Ngo, J.T. and Tirrell, D.A. (2011) Noncanonical amino acids in the interrogation of cellular protein synthesis. Accounts Chem. Res. 44, 677–685